The present invention relates to monoclonal antibodies and antigens, and to the use of such antibodies and antigens in the detection of the presence and concentration of certain specific microorganisms implicated in the etiology of human periodontal diseases. More specifically, the present invention relates to monoclonal antibodies specific to antigens from Actinobacillus actinomycetemcomitans. Actinobacillus actinomycetemcomitans is generally associated with juvenile periodontitis.
Clinical assays specific to such bacteria in gingival and subgingival dental plaque are useful in the diagnosis of periodontal disease, in evaluating the progress of periodontal therapy, and in determining the status of the patient at subsequent recall examinations. The standard bacteriological techniques presently used to identify such microorganisms are time consuming, expensive, and require a high level of expertise. Further, such tests frequently give results which are not as accurate or as sensitive as desired or required.
Recently, various efforts and proposals have been made to improve or replace standard bacteriological techniques by the utilization of immunodiagnostic assay techniques. Such immuno assay techniques are based upon the formation of a complex between the antigenic substance being assayed and an antibody or antibodies in which one or the other member of the complex is labeled. For example, labeling may be by means of a radioactive element, such as I.sup.125, (radioimmunoprecipitation assays); a material having fluorescent properties (immunofluorescence assays); enzyme-linked immunoabsorbant assays (ELISA) or (immunoperoxidase assays). The labeled member of the complex facilitates detection and/or qualitative analysis of the complexed labeled antigen or antibody from the uncomplexed antigen or antibody.
In typical competition utilizing immunoassay assay techniques, the antigenic substance in the sample of the fluid being tested competes with a known quantity of labeled antigen for a limited quantity of antibody binding sites. Thus, the amount of labeled antigen bound to the antibody is inversely proportional to the amount of antigen in the sample. In contrast, typical immunometric assays employ a labeled antibody. In such assay, he amount of labeled antibody associated with the complex is directly proportional to the amount of antigenic substance in the fluid sample.
Although immunodiagnostic techniques can be a substantial improvement over previously used bacteriological detection techniques, many further improvements remain to be made. For example, the immunodiagnostic techniques presently in use frequently produce variable results because relatively crude antigens and antibodies are employed. For example, three serotypes of Actinobacillus actinomycetemcomitans are known to exist in the oral cavity of man, yet one or more of the serotypes frequently goes undetected using present immunodiagnostic techniques. Similarly, there are at least three serologic variants of B. gingivalis in the oral cavity of man and they differ in virulence, hence the identification of the serologic variants of B. gingivalis by immunologic means can offer considerable advantages over other methods of detection and quantitations, however, present methods do not allow such distinction to be made. The immunodiagnostic techniques which are currently available for use in the determination of periodontal diseases generally lack accuracy, sensitivity and specificity.
Monoclonal antibodies may be defined as identical proteins produced by known techniques. The basic techniques for producing monoclonal antibodies were developed in the mid-1970's after Kohler and Milstein successfully fused spleen lymphocytes with malignant cells (myelomas) of bone marrow primary tumors [C. Milstein, Sci Am. 243(4): 66-74, (1980)]. Monoclonal antibodies recognize a single specific antigenic determinant (chemical structure). A given antigen may have several determinants. Thus, if monoclonal antibodies alone are utilized, immunodiagnostic tests may lack specificity because the same determinant may be found on different antigens or molecules, or false reading may be made because a single antigen may contain repeating determinants. Conventionally produced antigens typically have multiple determinants. Until the present invention, efforts to solve this problem have been made without a great deal of success. It is highly desirable that monoclonal antibodies be produced that not only have the ability to recognize a desired molecule or structure in a complex, but also have the ability not to recognize the same determinant in an unrelated or undesired complex. The present invention provide a specific antigen for microorganisms which are associated with periodontal diseases and to monoclonal antibodies that recognize only those antigens, and more particularly to monoclonal antibodies that recognize specific species, or strains, of such antigens. Thus the present invention provides a means of substantially improving the accuracy of various immuno-diagnostic assays.